转染RAW264.7细胞用荧光素酶报告基因
Summary
Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell’s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.
Abstract
Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.
Introduction
核酸细胞转染在科研多样的应用程序。实例包括（1）的报告基因来研究不同基因元件在基因表达中的作用;（2）蛋白表达质粒过表达感兴趣的蛋白质，和（3）的小干扰RNA下调基因表达。通过操纵特定基因的表达水平和测量这种操作的微分效果，研究人员可以推导出在所选择的生物系统的基因功能。不是所有的转染方法提供相同的转染效率，甚至同一染方法不转染所有类型的细胞同等1。因此，不同的转染方法已被开发，如磷酸钙法，DEAE-葡聚糖，阳离子脂质转染，阳离子非脂质的聚合物的转染，电穿孔，核转染和2,3。
转染巨噬细胞是ESPEcially困难的，由于这一事实，即巨噬细胞是专业的吞噬细胞是给外国的材料，包括来自（甲基）的DNA 4菌非常敏感。外源DNA的引入激活Toll样受体9（TLR9）途径，导致细胞因子的产生和一氧化氮5,6。然后，这些活化巨噬细胞可能是不太适应的治疗，研究人员打算检查。
我们的实验室常规transfects与荧光素酶报告基因的RAW264.7巨噬细胞系，我们已经开发了一个协议，是足够强大的荧光素酶有比信号背景显著高，而且柔情似水的巨噬细胞，以保持其静止状态。转染的细胞的行为由萤火虫荧光素酶报告基因窝藏IκBζ（pGL3-IκBζ）的启动子区进行了评价。 IκBζ表达通过细菌细胞壁成分唇上调opolyssacharide（LPS）的7,8，和由抗炎细胞因子下调，白介素-10（IL-10）8。考虑到井之间转变体中，我们共同转染含有海肾荧光素酶基因（ 例如，phRL-TK）进行归一化目的的对照质粒。描述的协议测试各种参数，包括转染的定时，对转染试剂的类型，转染试剂和质粒DNA的量，以及转染试剂的比率，以质粒DNA后进行了优化。包括在这个协议在两个转染试剂是：（1）一个基于脂质的转染试剂和（2）一种蛋白质/聚胺系转染试剂。
Protocol
Representative Results
Discussion
在这里描述的协议并不仅仅着眼于转染效率，但旨在在效率的细胞的生理状态和保护之间的平衡。具体地讲，我们的过程成功地减少转染试剂的毒性和最大化的荧光素酶信号。
在协议中的一个关键步骤是在细胞的健康。杂草丛生文化不适合的转染作为其生理变化，RAW264.7细胞的一个长的时间内还可以改变单电池10的表型和功能的连续培养。刚解冻的细胞，具有低传代?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This study was funded by Canadian Institutes for Health Research (CIHR) grant. STC holds a doctoral research award from the CIHR and the Michael Smith Foundation. EYS holds a CIHR scholarship. The CIHR Transplantation Training Program also provided graduate scholarships to STC, EYS and SS.
Materials
| PureLink HiPure Plasmid Maxiprep Kit | Life Technologies | K210007 | Any maxiprep kit will work |
| Phenol:chloroform:isoamyl alcohol | Life Technologies | 15593-049 | Molecular Biology Grade. Phenol is toxic so work in the fume hood, if possible. Use the lower clear organic layer if two layers of liquid form in the container. |
| DMEM | Thermo Scientific | SH30243.01 | Warm in 37°C water bath before use. |
| Fetal Bovine Serum | Thermo Scientific | SH30396.03 | Inactivated at 56°C water bath for 45 minutes before use. |
| Opti-MEM | Life Technologies | 31985-070 | Warm to at least room temperature before use. |
| XtremeGene HP DBA transfection reagent | Roche | 6366236001 | Warm to room temperature before use. |
| GeneJuice | EMD Millipore | 70967 | Warm to room temperature before use. |
| 5X Passive Lysis Buffer | Promega | E1941 | 30 ml is included in the Dual Luciferase Reporter Assay System |
| Dual Luciferase Reporter Assay System | Promega | E1910 |
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